Heparin-induced thrombocytopenia/thrombosis (HIT/T) is the most frequent cause of drug-induced antibody-mediated thrombocytopenia, a common cause of life-and limb-threatening platelet activation and thrombosis, and a model to study how an exogenous molecule (heparin) induces antibody formation against a self protein (platelet factor 4; PF4). Although antibodies to heparin/PF4 complexes are found in essentially all patients with HIT/T, it is unclear how antibody formation is initiated, why only a small subset of these antibodies cause disease or the mechanism by which they initiate thrombosis. We have recently developed a double transgenic mouse model and showed in vivo that heparin, PF4, antibodies to the heparin/PF4 complex, and the platelet Fc receptor for IgG, FcgammaRIIA are both necessary and sufficient to recapitulate the severe thrombocytopenia and thrombosis seen in patients with HITT. We have also found that PF4 and heparin form large oligomeric complexes that are immunogenic in vitro and are recognized by HIT antibodies. We will use these new findings to explore the immune mechanisms of antibody development, antigen recognition and pathogenesis of thrombosis in HIT through three interrelated aims. In Specific Aim 1 we will determine the heparin:PF4 ratio that supports the development of thrombocytopenia and thrombosis in vivo using mice in which the endogenous mouse PF4 has been knocked out. In Specific Aim 2 we will examine the structure of heparin/PF4 oligomers, their recognition by HIT antibodies, molecular composition, capacity to promote antibody-mediated platelet activation through FcgRIIA, and uptake and processing by dendritic cells. We will develop a library of human IgG1 and IgG2 antibodies to these complexes in XenoMouseII mice. In Specific Aim 3 we will systematically vary four host factors important to HIT/T development in vivo, in order to determine their contribution to the spectrum of human disease. The antibody source and titer, the heparin:PF4 structure, Fcg receptor variants that influence the balance of platelet activation and splenic clearance, and atherosclerotic vasculature will be examined in genetically-defined mouse models. Taken together these studies will provide insight into the development of self-reactive antibodies and thrombosis in patients with HIT/T, facilitate development of new diagnostic tests to identify patients at risk, provide a platform to design rational inhibitors of antibody development and provide a model to test new therapies for this serious disease.